DESCRIPTION: The information from the sequencing and annotation of the Human Genome has greatly facilitated basic research into the fundamental mechanisms involved in human biology and has been critical for identifying novel molecular targets to discover new therapies to treat common diseases as well as diseases that were previously untreatable. Our goal is to move functional genomics and proteomics into the 21st century by discovering new functions of old genes and identifying unique physiology for genes for which we have limited or no knowledge of their function. The work will focus on identifying functions of two important subfamilies of the Druggable Genome, the G protein-coupled receptor (GPCR) and nuclear hormone receptor (NHR) genes but will be adaptable to reveal unique activities of almost any gene, including protein kinases and ion channels. We will develop a novel technology platform that can measure a large array of different cellular functions of an individual gene, simultaneously, in living differentiated human neurons. The platform is based on robotic microscopy (RM), an automated live-cell imaging technology that monitors longitudinal single-cell functions that are analyzed statistically as in clinical trials. An innovation of RM is that i is 100-1000-fold more sensitive than any commercial system. Using RM and human induced pluripotent stem cell (iPSC)-derived neurons, we will develop an integrated, massively parallel approach to identify the functional capabilities of most human genes. This will be done using fluorescent biosensors we previously developed to assess hundreds of functions and pathways in neurons, including electrical activity and Ca++ mobilization, gene expression, flux through biological pathways (e.g., autophagy and proteasome), organelle morphology, and microtubule-based transport. These developed assays will be integrated with new ones to measure protein and organelle trafficking, gene transcription and translation, as well as other cell functions, int a single high-throughput 384- well format that can measure each response simultaneously and which will be analyzed using novel software. This platform can define the cellular physiological profile of a gene over seconds to days, without prior knowledge of that gene's function and will be developed for use on human neurons as well as other human cell types to identify functions of any gene. The innovation of this proposal is that we will develop a physical exam of the cell to identify functions of both known and orphan GPCRs and NHR. In the same way that the physical exam in medicine enables a physician to rapidly narrow down a universe of possibilities and hone in on critical physiology to be explored in depth, we have chosen an array of biosensors to scan diverse cell physiology to hone in on the critical functions of the target gene in question. We focus, but not exclusively, on human neurons because most of the druggable genome is expressed in the brain, and most drugs approved by FDA target gene products expressed in brain. Revealing novel functions of brain genes may provide the basis for developing a new generation of drugs to treat CNS diseases for which no effective treatments are available.